Generation of a Deep Mouse Brain Spectral Library for Transmembrane Proteome Profiling in Mental Disease Models

Transmembrane (TM) proteins constitute over 30% of the mammalian proteome and play essential roles in mediating cell-cell communication, synaptic transmission, and plasticity in the central nervous system. Many of these proteins, especially the G protein–coupled receptors (GPCRs), are validated or candidate drug targets for therapeutic development for mental diseases, yet their expression profiles are underrepresented in most global proteomic studies. Herein, we establish a brain TM protein-enriched spectral library based on 136 data-dependent acquisition runs acquired from various brain regions of both naïve mice and mental disease models. This spectral library comprises 3043 TM proteins including 171 GPCRs, 231 ion channels, and 598 transporters. Leveraging this library, we analyzed the data-independent acquisition data from different brain regions of two mouse models exhibiting depression- or anxiety-like behaviors. By integrating multiple informatics workflows and library sources, our study significantly expanded the mental stress-perturbed TM proteome landscape, from which a new GPCR regulator of depression was verified by in vivo pharmacological testing. In summary, we provide a high-quality mouse brain TM protein spectral library to largely increase the TM proteome coverage in specific brain regions, which would catalyze the discovery of new potential drug targets for the treatment of mental disorders.


Legends for Supplemental Figures
Legends for Supplemental Tables Table S1.Protein quantity, peptide numbers and protein coverage in the depression model with three library-based workflows Table S2.Protein quantity, peptide numbers and protein coverage in the anxiety model with three library-based workflows Table S3.DE TM proteins identified in the depression or anxiety model with three library-based workflows Table S4.References for disclosed regulators of depression or anxiety Table S5.Variable DIA windows

Figure S1 .
Figure S1.Comparison of three spectral libraries Figure S2.Proteome identification and quantification performance in the depression model data analysis with two library-free workflows Figure S3.Comparison of DE TM proteins quantified in three regions Figure S4.Comparison of DE TM proteome in depression model vs anxiety model Figure S5.Comparison of TM proteome regulation in depression model vs anxiety model analyzed by the SN-HB workflow Figure S6.Behavior tests of prosaptide TX14(A) in naïve mice Figure S7.Behavior tests of mice treated by ketamine or prosaptide TX14(A)

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Fig. S1.Comparison of three spectral libraries.A, Number of proteins from FragPipe library and the DDA library.DDA library is from our previous work.B, Venn plots of total proteins, TM proteins, GPCRs, ion channels and transporters from our three libraries.

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Fig. S2.Proteome identification and quantification performance in the depression model data analysis with two library-free workflows.A, Average number of protein IDs in each brain region with two analysis workflows (DIA-NN-Lib free, DIA-NN with library-free mode; SN-directDIA, Spectronaut with directDIA mode).B, Comparison of the total proteins, TM proteins or GPCRs reported by two analysis workflows.C, CV distribution of TM proteins from specific brain regions, indicating the high reproducibility of two library-free workflows.D, Missing values for both library-based and library-free workflows.

Fig. S3 .
Fig. S3.Comparison of DE TM proteins quantified in three regions.Venn plots of DE TM proteins discovered by three workflows in depression model (A) and anxiety model (B).

Fig. S4 .
Fig. S4.Comparison of DE TM proteome in depression model vs anxiety model.Venn plots of DE TM and GPCR proteins in three regions of two models.

Fig. S5 .
Fig. S5.Comparison of TM proteome regulation in depression model vs anxiety model analyzed by the SN-HB workflow.A, Number of total TM proteins quantified in three regions of depression and anxiety models.B, Venn plots showing the overlap of the TM proteins profiled in two mouse models.C, Venn plots of DE TM or GPCR proteins from three regions of two models.D, PCA plot of three regional expression of total TM proteins demonstrates the clustering of control, depression or anxiety groups, with distinct separation of two models.E, Significantly enriched cell types (adjusted p <0.05) of DE TM proteins from two models.F, Significantly enriched pathways (adjusted p <0.01) of up-or down-regulated TM proteins from the depression model.No significant enriched pathways are obtained for anxiety model.OPCs, Oligodendrocyte Progenitor Cells; Retrograde EC signaling, Retrograde endocannabinoid signaling; Pathways of NDM diseases, Pathways of neurodegeneration − multiple diseases

Fig. S7 .
Fig. S7.Behavior tests of mice treated by ketamine or prosaptide TX14(A).A, No effect was observed for ketamine at 24 h post-infusion as measured in OFT.B, Antidepressant effects of stereotactic infusion of ketamine at 1 hour and 24 hours postinfusion as measured in FST.C, D, No antidepressant effects of stereotactic infusion of prosaptide TX14(A) (1.25 and 5 μg each side) into the mPFC at 24 h (C) or 3 days (D) post-infusion as measured in the OFT (C), TST (D) and SPT (D).Data are mean ± SEM, ****p <0.0001.